The induced water activity effect upon intercalation of actinomycin D (actD) to DNA has been studied using osmotic stress methods. The binding isotherms and constants obtained by spectrophotometric titration indicate that solutes which are excluded from the drug and DNA surfaces induce higher binding levels upon complex formation. These measurements have also revealed a second class of binding sites which are not accessible in the absence of co-solvents and vary linearly with water activity when plotted on logarithmic scales. From the latter, we have determined the number of water molecules released upon drug binding ( nw) which is proportional to the co-solvent molecular weights or molecular volumes. This linkage between the hydration and free energy changes on binding with solute molecular volumes suggests that the entropic gain upon releasing nw to the bulk solvent upon drug binding is a source for the higher ActD intercalation levels observed in the presence of these co-solvents. At moderate ionic strength and in the absence of co-solvents, ActD intercalation presents an unfavorable enthalpy contribution ( HvH = +1.8 kcal/mol). Our hypothesis is that the entropic contributions resulting from the exclusion of the co-solvent could compensate for this positive enthalpy resulting in more favorable the binding energetically more favorable. To test this hypothesis, we have measured the calorimetric and van't Hoff enthalpy of interacalation in the presence of different concentrations of sucrose. Preliminary measurements indicate that HvH decreases linearly with an increase in sucrose concentration and a resultant increase in entropy of dissolving nw water molecules. For example, the HvH measured in 30% (W/V) sucrose equals - 8.0 kcal/mol, a value that is usually observed for the intercalation of other drugs to DNA. Calorimetric measurements of ActD binding to DNA at low r ratios in the absence and presence of sucrose are consistent with the spectroscopically derived van't Hoff enthalpies. These data emphasize the importance of water and suggest that non-ideality of the cellular medium exerts significant control on the affinity of drug-DNA interactions.